Recombinant Human FAM20C Protein, CF

• 6 months from date of receipt, -20 to -70 °C as supplied.
• 3 months, -20 to -70 °C under sterile conditions after opening.

• Universal Kinase Activity Kit (Catalog # EA004)
• Assay Buffer: 25 mM HEPES, 150 mM NaCl, 10 mM MnCl₂, 10 mM CaCl₂, pH 7.0
• Recombinant Human FAM20C (rhFAM20C) (Catalog # 9265-FM)
• 96-well Clear Plate (Catalog # DY990)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute 1 mM Phosphate Standard provided by the Universal Kinase Activity Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
2. Complete the standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock using Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
3. Dilute ATP (supplied in kit) to 0.4 mM in Assay Buffer.
4. Dilute rhFAM20C to 33.34 ng/µL in Assay Buffer.
5. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
6. Load 15 µL of 33.34 ng/µL rhFAM20C into empty wells of the same plate as the curve. Include a Control containing 15 μL of Assay Buffer.
7. Add 25 µL of 0.4 mM ATP into all wells, excluding the standard curve. 
8. Seal plate and incubate at 37 °C for 1 hour.
9. Dilute Coupling Phosphatase 4 (supplied in kit) to 10 ng/µL in Assay Buffer.
10. Add 10 µL of 10 ng/µL Coupling Phosphatase 4 to all wells, excluding the standard curve.
11. Seal and incubate plate at room temperature for 5 minutes.
12. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
13. Add 100 µL of deionized water to all wells. Mix briefly.
14. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate sealed plate for 20 minutes at room temperature.
15. Read plate at 620 nm (absorbance) in endpoint mode.
16. Calculate specific activity:

     *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.
     ** Based upon a 65 minute incubation time.  (No coupling rate constant.) 

Per Reaction:

• rhFAM20C: 0.5 µg
• Coupling Phosphatase 4: 0.1 µg
• ATP: 0.2 mM